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Use of Magnetic Beads for Plasma Cell-free DNA Extraction: Toward Automation of Plasma DNA Analysis for Molecular Diagnostics

¹ Service de Biochimie et Biologie Moléculaire, Hôpital de Hautepierre Hôpitaux Universitaires de Strasbourg, 67000 Strasbourg, France;
² Service de Chirugie Urologique and
³ Service de Pneumologie Lyautey, Hôpitaux Universitaires de Strasbourg, 67000 Strasbourg, France

^aaddress correspondence to this author at: Service de Biochimie et Biologie Moléculaire, Hôpital de Hautepierre, Hôpitaux Universitaires de Strasbourg, 67098 Strasbourg cedex, France; fax 33-388127539, e-mail christine.stemmer@wanadoo.fr

The first 300 words of the full text of this article appear below.

Urine, breast milk, plasma, and serum have been shown to contain cell-free DNA (1)(2)(3)(4)(5)(6)(7). For plasma DNA detection, several recent studies addressed the need for careful evaluation and standardization of preanalytical processes (8)(9)(10)(11)(12). Key problems appear, such as possible contamination of plasma by white blood cells; the generally low and variable amount of circulating DNA, making extraction/quantification difficult and time-consuming; poor DNA quality; and the presence of PCR inhibitors. In any case, automation of DNA extraction, which is a prerequisite for introduction of these diagnostic approaches in clinical laboratories, is difficult to achieve because of the volumes of plasma necessary to get sufficient DNA.

In this study (summarized in Fig. 1 ), we propose a new semiautomated, time-saving process for extraction of plasma cell-free DNA that provides high yields and is suitable for PCR amplification.

View larger version (34K): [in this window] [in a new window]   Figure 1. Experimental design. Step 1, plasma treatment; step 2, DNA extraction; step 3, analysis of plasma DNA quality by PCR.

Two 5-mL blood samples from each of 23 patients being monitored for lung (n = 19) or colon cancer (n = 4) and 20 healthy controls were collected in EDTA-containing tubes. Informed consent was obtained for each. Blood was centrifuged at 800g for 10 min, and the collected plasma was transferred to a 15-mL BD Falcon^TM polypropylene tube for an additional centrifugation step of 10 min at 1500g to remove any remaining leukocytes and platelets. An equal volume of 2x concentrated proteolytic buffer (2x: 20 mmol/L Tris-HCl, 50 mmol/L EDTA, 200 mmol/L NaCl, 10 g/L sodium dodecyl sulfate, and 400 mg/L proteinase K) was added to the plasma, mixed, and incubated 1 h at . . . [Full Text of this Article]

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